báo cáo khoa học: " UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures" pps

BioMed Central Page 1 of 17 (page number not for citation purposes) BMC Plant Biology Open Access Research article UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures Shilpa Ramani and Jayabaskaran Chelliah* Address: Department of Biochemistry, Indian Institute of Science, Bangalore, 560012, India Email: Shilpa Ramani - shilpasuhas@gmail.com; Jayabaskaran Chelliah* - cjb@biochem.iisc.ernet.in * Corresponding author Abstract Background: Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor- signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc) and strictosidine synthase (Str). In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results: Here, we investigate whether cell surface receptors, medium alkalinization, Ca 2+ influx, H 2 O 2 , CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion: Our results demonstrate that cell surface receptor(s), Ca 2+ influx, medium alkalinization, CDPK, H 2 O 2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed. Background C. roseus produces terpenoid indole alkaloids (TIAs) as a part of its secondary metabolism. TIAs provide protection against microbial infection, herbivores and abiotic envi- ronmental stresses such as UV irradiation [1,2]. Some of the TIAs are of pharmaceutical importance such as the antitumor dimeric alkaloids, vincristine and vinblastine, and the anti-hypertensive monomeric alkaloids, ajmali- cine and serpentine [3]. The anti-tumor dimeric alkaloids, which accumulate in the leaves of C. roseus, are composed of catharanthine and vindoline monomers and are exclu- sively found in C. roseus plants. In plants, the dimeric alka- loids and the monomer catharanthine accumulate in low amounts whereas the monomer vindoline accumulates at Published: 7 November 2007 BMC Plant Biology 2007, 7:61 doi:10.1186/1471-2229-7-61 Received: 13 November 2006 Accepted: 7 November 2007 This article is available from: http://www.biomedcentral.com/1471-2229/7/61 © 2007 Ramani and Chelliah; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 2 of 17 (page number not for citation purposes) a relatively higher level [4,5]. C. roseus cell cultures have been investigated as alternative means of production of terpenoid indole alkaloids, but they failed to produce vin- doline [6]. Therefore, it has been considered desirable to produce the dimers by coupling catharanthine obtained from cell cultures with vindoline obtained from the culti- vated plants. The production of catharanthine by C. roseus cell cultures has been one of the most extensively explored areas of plant cell culture and is still limited due to the low yield [7]. Elicitations are considered to be an important strategy towards improved in vitro production of secondary metab- olites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites [8]. Fungal elicitors have been widely tested for elicitation of catharanthine production in various C. roseus cells [5,9]. However, molecular basis of elicitor-sig- naling cascades leading to increased production of sec- ondary metabolites of plant cell is largely unknown. It is known that receptor proteins that bind elicitors generate signals that are transmitted to the sites of gene expression via different components, such as Ca 2+ /ion fluxes, medium alkalinization and cytoplasmic acidification, oxi- dative burst, jasmonate and nitric oxide etc. [8]. Many CDPKs and MAPKs have been identified to play a role in defense responses and also secondary metabolite produc- tion [10]. The effect of UV-B irradiation on expression of TIA biosyn- thetic genes, Tdc and Str, and catharanthine production has been reported previously in C. roseus leaves[11-13]. The transcription factor GT-1 binds to the promoter region of Tdc in vitro. The functional importance of GT-1 in the induction of Tdc expression by UV light has been demonstrated by point mutations in the GT-1 binding site [14]. However, the molecular basis of UV-B signaling cas- cades leading to the induction of expression of Tdc and Str genes and the production of TIAs is largely unknown. It has been observed that the polypeptide wound signal, sys- temin- specific cell surface receptors initiate a signal trans- duction cascade upon UV-B irradiation in L. peruvianum cell suspension cultures [15]. In the present study, the sig- naling pathways mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were inves- tigated. UV-B induced alkalinization of the culture medium, generation of hydrogen peroxide, activation of CDPK and MBPK as well as accumulation of catharan- thine and stimulation of transcription of Tdc and Str genes were studied. Inhibitors of binding of ligand-cell surface receptors, protein kinases and phosphatases, calcium fluxes and H 2 O 2 were used to dissect the UV-B signaling cascade. Results Alkalinization of C. roseus cell-suspension medium in response to UV-B irradiation and its inhibition by suramin Medium alkalinization an early event occurring in elici- tor- treated plant cell cultures, has been used as a marker of elicitor responses in studying elicitor-binding sites in plant cells [16]. Medium alkalinization is thought to result from elicitor/stress-induced depolarization of the plasma membrane and subsequent K + /H + exchange with Ca 2+ influx/Cl - efflux [16]. To determine whether medium alkalinization is involved in UV-B signal transduction as an early event, six-day-old cells were exposed to UV-B irra- diation for various time periods (2, 5, 10 or 20 min) and extracellular pH changes were measured in the cell-sus- pension medium for 120 min. As shown in Figure 1a, the effect of UV-B on medium alkalinization was not dose- dependent. However, the kinetics and intensity of this response were dependent on their respective exposure times. C. roseus cells showed a rapid increase in the medium pH after UV-B irradiation peaking at 10 min with an increase of about 0.7 units in 5-min irradiated cells (Fig 1a inset). The other doses of UV-B irradiation on cells did cause an increase in AR, but in all cases the pH of the medium decreased but never returned back to baseline levels even after 24 h, which probably could be due to the damage caused by prolonged exposure to UV-B (data not shown). In the cells irradiated with 2 and 5 min of UV-B however, the pH of the medium returned to baseline by 300 min (data not shown). Cell viability when checked after 24 h of irradiation showed that irradiation with UV- B for 2 min and 5 min did not cause cell death (98% cell survival as visualized by florescein diacetate/propidium iodide staining); however, irradiation for longer than 5 min caused 80 – 100 % cell death (data not shown). We have therefore used 5 min of UV-B as the standard irradi- ation time for all further experiments. Suramin is known to bind with cell surface components such as the systemin receptor [17] and interfere with the signaling events and this system is affected by UV-B irradi- ation in L. peruvianum cells [15]. Since UV-B irradiation of C. roseus cells caused alkalinization of the medium, we investigated whether suramin could inhibit the UV-B- induced medium alkalinization. The results show that the UV-B-induced alkalinization was inhibited by suramin (Figure 1b). Suramin inhibited alkalinization of the growth medium for all exposure times of UV-B irradia- tion. Heparin, which is similar to suramin in possessing polysulfonated groups, had no effect on alkalinization of the medium induced by UV-B irradiation. UV-B-induced H 2 O 2 production and involvement of protein kinases in UV-B-induced H 2 O 2 production The oxidative burst, a rapid consumption of oxygen and production of reactive oxygen species (ROS) such as BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 3 of 17 (page number not for citation purposes) H 2 O 2 , is a typical early event in plant defense responses [18,19]. With 5 min of UV-B irradiation of C. roseus cells H 2 O 2 production increased six-fold compared to control cells (Fig 2a). We next examined effects of suramin, an inhibitor of G-protein inhibitor, N-acetyl cysteine, a puta- tive ROS scavanger, verapamil, a calcium channel blocker and staurosporine, a serine-threonine kinase inhibitor, SB 203580, a P38 MAPK inhibitor, PD 98059, an ERKK inhibitor and SB 600125 JNK inhibitor. The UV-B- induced H 2 O 2 production was suppressed by all the inhibitors except the MAPK cascade inhibitors (Fig 2b). This indicated that upon receiving the UV-B signal by a putative receptor in C. roseus cells, calcium influx and acti- vation of serine/threoine kinases are required to induce H 2 O 2 production. However, activation of the MAPK cas- cade occurs downstream of H 2 O 2 production. Activation of protein kinases in response to UV-B irradiation in C. roseus suspension cell cultures Many protein kinases are known to respond to both biotic and abiotic stresses. Two kinases, MAPKs and CDPKs, have been implicated to play pivotal roles in response to diverse stimuli [17,20]. Previous studies have demon- strated that C. roseus cells also respond to UV-B irradiation by expressing biosynthetic genes and production of TIAs [13]. To establish a functional link between these proc- esses, we first examined the possible activation of MAPK and CDPK in cells irradiated with UV-B. MBP is known to be a conventional MAPK substrate and MAPK homologs also have MBP kinase activity [21]. To determine if a MAPK is associated with the UV-B signaling the activation of MBP kinase was investigated C. roseus cell suspensions were exposed to UV-B irradia- tion for 5 min and the cells were then assayed for MBPK and CDPK activities for different time periods. In vitro assays were performed in the cell extracts prepared from UV-B irradiated and control C. roseus cells. Figure 3a indi- cates that MBPK activity in UV-B irradiated cells signifi- cantly increased by 5 min and peaked at 10 min after UV- B irradiation. The MBPK activity remained high and above the control levels even at 20 min following irradiation. In order to identify specific MBPK activity induced by UV-B, an in-gel kinase assay was carried out. Figure 3b shows that in UV-B irradiated cells, the activity of one major pro- tein kinase could be detected in the polyacrylamide gel containing MBP. From the mobility of the MBPK activity band during SDS-PAGE, the apparent molecular mass of Medium alkalinization of C. roseus suspension cultured cells in response to UV-B irradiation and its inhibition by suraminFigure 1 Medium alkalinization of C. roseus suspension cultured cells in response to UV-B irradiation and its inhibition by suramin. (a)Six- day-old cell suspension cultures were either irradiated with UV-B or left untreated for various periods of time and the pH of the medium was measured at the times indicated after the start of irradiation. Alkalinization response (AR or ∆ pH) was meas- ured as described in materials and methods. Inset: Early medium alkalinization response to 5 min of UV-B irradiation (b) Inhibi- tion by suramin of UV-B-induced medium alkalinization. Cells were pre-treated with 1 mM suramin or 1 mM heparin for 10 min prior to irradiation with different doses of UV-B, and as control, cells were irradiated with UV-B alone and the pH of the medium was measured after 10 min. The increase in medium pH (∆ pH) is indicated as the difference between the pH at time 0 and at 10 min. Bars represent the means ± SD (n = 6). BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 4 of 17 (page number not for citation purposes) the enzyme was estimated to be approximately 49 kDa. The 49-kDa MBPK activity increased by UV-B irradiation in cells compared with that of the un-irradiated control. The maximum MBPK activity was observed at 10 min after UV-B treatment. In all the in vitro experiments carried out with MBP as substrate, the phosphorylation peaked at 10 min; these results were consistently obtained when the experiments were repeated with different batches of cells. Therefore, in all further experiments the MBPK activity was assayed at 10 min after irradiation. To further characterize the MBPK activity induced by UV- B, immunoprecipitation and in-gel kinase assays were used. The protein extracts were incubated with anti-phos- photyrosine monoclonal antibody and immunoprecipi- tated with protein A-agarose. The immunoprecipitated proteins were separated on a SDS-polyacrylamide gel con- taining MBP as a substrate and MBPK activity was assayed in the gel in the presence of 32 P- ATP. As shown in Figure 3c, a 49 kDa protein kinase was again detected in the immunoprecipitate from UV-B-irradiated cells. Co-incu- bation with phosphotyrosine prevented immunoprecipi- tation of the 49 kDa protein kinase with anti- phosphotyrosine antibody, but co-incubation with phos- phothreonine did not. These results indicate that only phosphotyrosine and not phosphothreonine could act as a competitor during immunoprecipitation, showing that MBP phosphorylating kinase was specifically phosphor- ylated on a tyrosine residue. Till date MAPK are the only known plant kinases to be phosphorylated on tyrosine residues. Calcium dependent protein kinases (CDPKs) belong to the unique family of calcium-regulated kinases and his- tone IIIS was one of the best exogenous substrates for assaying CDPKs [22]. To characterize the kinase(s) induced by UV-B, the activities were assayed using histone IIIS as a substrate in protein extracts from cells irradiated with UV-B, as well as the controls. The protein extracts from 5-min UV-B irradiated cells, assayed in the presence of calcium using histone IIIS as substrate showed that, the kinase activity increased significantly peaking at 4 min after UV-B irradiation and remained high even at 20 min after UV-B irradiation (Figure 4a). The protein extracts from 5-min UV-B irradiated cells assayed by in- gel kinase assay in the absence and presence of calcium using his- tone IIIS as substrate demonstrated that the phosphoryla- tion of histone IIIS was calcium dependent in both UV-B irradiated and un-irradiated cells (Figure 4b). CDPK activ- ities were identified at two positions with an apparent molecular weight of 55 kDa and 40 kDa. One of the CDPK activated had an apparent molecular weight of 40 kDa and was constitutive, as it was observed to phospho- rylate histone IIIS to a similar extent in both un-irradiated and irradiated cells whereas the 55 kDa kinase activity showed UV-B dependence and peaked at 4 min. There- fore, the phosphorylation of histone IIIS observed in vitro experiments was both due to the activities of the 55 and Production of ROS in C. roseus suspension cultured cells in response to UV-B irradiationFigure 2 Production of ROS in C. roseus suspension cultured cells in response to UV-B irradiation. (a) A time course of UV-B induced ROS production. Six-day-old cell suspension cultures were irradiated by UV-B for different times and 2.5 µM DCFH-DA was added. The ROS production was measured after 15 min as a difference in the fluorescence intensity between the UV-B-irradi- ated and untreated controls. Bars represent means ± SD (n = 3). (b) Effect of various inhibitors on UV-B induced ROS produc- tion. Six-day-old cell suspension cultures were treated with 1 mM suramin (Sur), 10 mM N-acetyl cysteine (NAC), 0.5 µM verapamil (Vera), 10 nM staurosporine (St), 40 nM SB 600125, a JNK inhibitor (SB6), 70 nM SB 203580, a P38 inhibitor (SB2) and 5 µM PD 98059, an ERKK inhibitor (PD) for 10 min prior to UV-B irradiation of 5 min and 2.5 µM DCFH-DA was added to the treated cultures. The ROS produced was measured as above. BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 5 of 17 (page number not for citation purposes) 40 kDa kinases. CDPKs being serine-threonine kinases are phosphorylated on both serine and threonine residues. To differentiate between MBP kinase detected in our experi- ments and the histone IIIS kinase, we used anti-phospho- serine monoclonal antibody for immunoprecipitation followed by a pull down with Protein A-agarose and Activation of Myelin Basic Protein Kinase (MBPK) activity by UV-B irradiation in C. roseus suspension cultured cellsFigure 3 Activation of Myelin Basic Protein Kinase (MBPK) activity by UV-B irradiation in C. roseus suspension cultured cells. Six-day-old cell suspension cultures were irradiated for 5 min with UV-B light (+) or left un-irradiated (-) as a control. Cells were har- vested at the indicated time periods, crude extracts were prepared, and MBPK activity in the cell extracts was assayed using MBP as a substrate as described in materials and methods. (a) MBPK activity was carried out with an in vitro phosphorylation assay. The reaction mixtures were resolved by SDS 10% (w/v) polyacrylamide gel electrophoresis and the phosphorylated MBP was visualized by autoradiography. (b) MBPK activity in the cell extracts was determined by in-gel kinase assay with MBP as a substrate. Autoradiogram represents in-gel phosphorylation of MBP. (c) Detection of MBPK activity in immunoprecipitates from cell extracts using the anti-phosphotyrosine antibody. Lane 1 and 2 represent cell extracts subjected to in-gel kinase assay directly without immunoprecipitation. Lane 3 to 10 indicate the cell extracts subjected to immunoprecipitation with a mono- clonal antibody specific for phosphotyrosine and the MBPK activity of the immunoprecipitates assayed by in-gel kinase assay. The phosphorylated MBP was visualized by autoradiography. Phosphotyrosine and phosphothreonine were used as competitor substrates to demonstrate the specificity of the antibody. Symbols (-) and (+) represent, untreated and treated of the indicated treatment. BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 6 of 17 (page number not for citation purposes) assayed by in-gel kinase assay containing histone IIIS as substrate. Figure 4c shows that the 55 and 40 kDa kinases identified by in-gel kinase assay in Figure 4b were both phosphorylated on serine residues and that the activity of 40 kDa kinase was constitutive in our cell cultures. In all the in vitro experiments carried out with histone IIIS as substrate, the phosphorylation peaked at 4 min. These results were consistently obtained when the experiments were repeated with different batches of cells. Therefore, in Activation of CDPK in C. roseus suspension cultured cells in response to UV-B irradiationFigure 4 Activation of CDPK in C. roseus suspension cultured cells in response to UV-B irradiation. Six-day-old cell suspension cultures were irradiated for 5 min with UV-B light (+) or left un-irradiated (-) as a control. Cells were harvested at the indicated time periods, crude extracts were prepared, and the activity of CDPK in the cell extracts was assayed using histone IIIS as a sub- strate as described in materials and methods. (a) CDPK was assayed with an in vitro phosphorylation assay. The reaction mix- tures were resolved by SDS 10% (w/v) polyacrylamide gel electrophoresis and subjected to autoradiography. (b) CDPK activity in the cell extracts were determined by in-gel kinase assay with histone IIIS as substrate in the presence and absence of calcium. Autoradiogram represents in-gel phosphorylation of histone IIIS. Arrows show the molecular masses of two detected CDPK bands (c) Detection of CDPK activity in immunoprecipitates from cell extracts using anti-phosphoserine antibody. Lane 1 and 2 represent cell extracts subjected to in-gel kinase assay directly without immunoprecipitation. Lane 3 to 7 indicate the cell extracts subjected to immunoprecipitation with a monoclonal antibody specific for phosphoserine and the CDPK activity of the immunoprecipitates assayed by in-gel kinase assay. The phosphorylated histone IIIS was visualized by autoradiography. Sym- bols (-) and (+) represent, untreated and treated of the indicated treatment. Arrows show the molecular masses of two detected CDPK bands. BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 7 of 17 (page number not for citation purposes) all further experiments the CDPK activity was assayed at 4 min after irradiation. UV-B-induced MBPK and CDPK activities, Tdc and Str gene expression and catharanthine accumulation are inhibited by suramin Since the UV-B-induced early cellular responses viz., medium alkalinization and ROS production were inhib- ited by suramin, we investigated whether suramin could inhibit the UV-B induced other cellular responses related to synthesis of TIAs. When the cells were pretreated for 10 min with 0.1 and 1 mM suramin concentrations and sub- sequently irradiated with UV-B for 5 min, the UV-B- induced MBPK and CDPK activities, accumulation of Tdc and Str transcripts and catharanthine was strongly inhib- ited (Figure 5a–d). However, the UV-B-induced MBPK activity could not be completely inhibited by suramin. To rule out the possibility that the inhibitory effects of suramin on responses triggered by UV-B are not due to the unspecific binding to cell surface components, we used heparin a structurally similar molecule viz., heparin that possesses sulfonic acid groups similar to that of suramin for inhibition of UV-B responses. Figure 5a–d shows that heparin at both 0.1 and 1 mM concentrations had no Effects of suramin and heparin on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseusFigure 5 Effects of suramin and heparin on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseus. Six-day-old cell suspension cultures were pre-treated with suramin (Sur) or heparin (Hep) at the indicated concentrations and were irradiated with UV-B for 5 min. As control one set of cells was irradiated with UV-B alone or left un-irradiated and the crude extracts from all cells were prepared at the indi- cated times and assayed for the phosphorylation of H IIIS (a) and MBP (b) under standard conditions as described in materials and methods. A second set of cells was similarly treated and the total RNA was isolated at the indicated times and analyzed for the transcript levels of Tdc and Str by RT-PCR (c). The third set of cells were pretreated with the highest concentration of inhibitor previously used followed by 5 min of UV-B irradiation. After treatment, cells were collected after 48 h and catharan- thine content was determined by HPLC (d). These experiments were performed in triplicates and repeated at least twice. Error bars represent mean ± SD (n = 3). BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 8 of 17 (page number not for citation purposes) effect on any of the UV-B mediated signaling events inves- tigated demonstrating that the effect of suramin was spe- cific under UV-B irradiated conditions. These data indicate that suramin-sensitive cell surface receptor may participate in the UV-B responses. Role of Ca 2+ in UV-B induced responses in C. roseus cells Changes in membrane permeability and the resulting ion fluxes mainly Ca 2+ and H + influx, and K + and Cl - efflux, are among the most rapid responses of plant cells to elicita- tion [23,24] Among these ion fluxes, the influx of Ca 2+ play an important role in transduction of the elictor signal and for elicitor-induced accumulation of plant secondary metabolites [25]. To assess whether Ca 2+ influx is involved in the UV-B-induced signaling pathway leading to catha- ranthine accumulation, the C. roseus cultured cells were treated with a specific calcium chelator EGTA prior to the UV-B irradiation and the UV-B induced responses were examined. Because EGTA is not likely to enter the cell, we expected it to make extracellular Ca 2+ at least partially unavailable for entering the cytoplasm by chelation. Pre- treatment with EGTA reduced the UV-B stimulated MBPK and CDPK activities to a very large extent indicating EGTA blocked the UV-B responses (Figure 6a and 6b). The level of the Tdc and Str transcripts and catharanthine content in the UV-B irradiated cells also reduced gradually as the EGTA concentration increased (Figure 6c and 6d). The involvement of calcium in the UV-B induced signaling pathway leading to catharanthine accumulation was fur- ther confirmed by studying the effect of verapamil, the plasma membrane calcium channel blocker, on the UV-B- induced responses. As shown in Figure 6a and 6b, vera- pamil inhibited the UV-B-induced MBPK and CDPK activ- ities to a significant extent. UV-B-induced accumulation of Tdc and Str transcripts also decreased upon treatment with verapamil (Figure 6c). The catharanthine content in vera- pamil pre-treated cells also reduced significantly (Figure 6d). These results indicate that UV-B-induced catharan- Effect of verapamil (Vera) and EGTA on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseusFigure 6 Effect of verapamil (Vera) and EGTA on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseus. Six-day-old cell suspension cultures were pre- incubated with verapamil or EGTA at concentrations indicated followed by 5 min of UV-B irradiation. Other details are as in the legend to Figure 5. BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 9 of 17 (page number not for citation purposes) thine accumulation requires elevated levels of cytosolic calcium, and this increase is brought about by an influx of calcium from extracellular space. Role of protein phosphorylation in UV-B induced responses in C. roseus cells Having established that the activation of a 49-kDa MBPK and 55-kDa CDPK was induced by UV-B irradiation of C. roseus cells (Figs 3 and 4), we used this property in combi- nation of inhibitors of protein kinases to assess possible involvement of these kinases in UV-B signaling pathway leading to catharanthine accumulation. The C. roseus cells were treated with inhibitors of protein kinases and the UV-B-induced responses, viz., MBPK and CDPK activities, Tdc and Str transcript accumulation and catharanthine content were examined. Staurosporine, a potent inhibitor of serine-threonine kinases, SB 203580, an inhibitor of P38 class of MAP kinase, PD 98059, an inhibitor ERKK class of MAPKK and SB 600125, an inhibitor of Janus kinases were used to assess the role of protein phosphor- ylation in UV-B responses. As shown in Figure 7a and 7b, staurosporine, SB 203580, PD 98059 and SB 600125 treatments at the concentrations tested completely abol- ished the UV-B-induced MBPK activity whereas the UV-B- induced CDPK activity could not be completely inhibited by staurosporine and was not inhibited by SB 203580, PD 98059 and SB 600125 pretreatments of the cells. The inhibitory effect of staurosporine on both MBPK and CDPK activities indicates a common mechanism of action of the inhibitor on these protein kinases, as both of them belong to the family of serine-threonine kinases. As expected, inhibitors of the MAPK cascade only inhibited the UV-B-induced MAPK-like MBPK activity, but not CDPK activity. We next examined the accumulation of Tdc and Str mRNA's in protein kinase inhibitor treated cells by reverse transcription polymerase chain reaction (RT-PCR). As shown in Figure 7c staurosporine, SB 203580, PD 98059 and SB 600125 inhibited UV-B-induced Tdc and Str transcript accumulation. In a similar fashion, UV-B- induced catharanthine production was significantly decreased by the above-mentioned inhibitors (Figure 7d) indicative of the implication of MBPK and CDPK activities in elicitation of UV-B induced catharanthine biosynthesis. The data obtained by immunoprecipitaion experiments and with the use of MAPK cascade specific inhibitors sug- gests the involvement of a putative MAPK in response to UV-B. As protein phosphatases antagonize the activity of protein kinases, we tested whether pre-treatment of cells with pro- tein phosphatase inhibitors would show the opposite effect on the UV-B-induced responses. Interestingly, the addition of orthovanadate, a known inhibitor of tyrosine phosphatases [26] or sodium fluoride, a compound reported to strongly inhibit serine-threonine phos- phatases [27], stimulated only the UV-B-induced MBPK activity at 1 and 10 mM concentrations substantially above the UV-B treated activity while that of CDPK activ- ity remained unaffected (Figure 8b and 8a). The pretreat- ment of cells with orthovandate and sodium fluoride did not substantially increase the CDPK activity over and above the UV-B treated cells. To further test the role of protein phosphatases in the UV-B-induced protein phos- phorylation activities, we used NAC, which is known to protect the thiol group of phosphatases from inactivation [26]. Pretreatment of cells with NAC inhibited the UV-B- induced MBPK and CDPK activities at 10 and 100 mM concentrations tested (Fig 8a and 8b). As shown in Figure 8c, pretreatment with orthovanadate or NaF did not increase the transcripts of Tdc and Str beyond the levels seen in cells irradiated with UV-B alone; however, NAC, on the other hand, decreased the UV-B-induced accumu- lation of Tdc and Str transcripts. At alkaloid level, we found that catharanthine accumulation in the C. roseus cells was greatly increased by UV-B irradiation (Figure 8d). Pretreatment of orthovanadate or sodium fluoride had no significant effect on the accumulation of catharan- thine over and above the cultured C. roseus cells irradiated with UV-B alone. NAC had an overall inhibitory effect on the UV-B-induced Tdc and Str transcript levels as well as the catharanthine accumulation. NAC apart from protect- ing phosphatases from inactivation is also a potent inhib- itor of ROS production. The results shown in Figure 2 as well as Figure 8 indicate that the UV-B signaling involves both ROS production and inactivation of phosphatases. Discussion Several studies have demonstrated the involvement of sig- nal components, such as receptors, Ca 2+ influx, medium alkalinization, oxidative burst, and protein kinases and phosphatases in responses to elicitors for enhanced pro- duction of secondary metabolites via increased transcrip- tion of relevant genes [8]. It has been shown earlier in C. roseus that the abiotic elicitor UV-B induces the formation of dimeric TIAs, and Tdc and Str mRNA accumulation [13]. There is also evidence that nuclear factor GT-1 func- tion in the regulation of Tdc gene expression by UV light in C. roseus [14]. However, the UV-B signaling pathway that regulates activity of transcription factor GT-1 leading to Tdc gene expression is still obscure. In the present study, we present evidence for involvement of a putative receptor(s), calcium, reactive oxygen species, Ca 2+ - dependent protein kinase, and a putative MAPK in UV-B signaling and transcriptional activation of Tdc and Str genes and catharanthine biosynthesis in C. roseus cells. Based on suramin interference with the binding of sys- temin to its cell surface receptor and UV-B responses in L. peruvianum cells [17] we used suramin to assess the involvement of a cell surface receptor in UV-B-induced BMC Plant Biology 2007, 7:61 http://www.biomedcentral.com/1471-2229/7/61 Page 10 of 17 (page number not for citation purposes) Effect of protein kinase inhibitor and MAPK cascade specific inhibitors on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseusFigure 7 Effect of protein kinase inhibitor and MAPK cascade specific inhibitors on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension cultures of C. roseus. Six-day-old cell suspension cultures were pre-incubated with staurosporine (St), SB 203580 a P38 inhibitor (SB2), PD 98059, an ERKK inhibitor (PD) or SB 600125 a JNK inhibitor (SB6) at concentrations indicated followed by 5 min of UV-B irradiation. Other details are as in the legend to Figure 5. [...]... thiolcatharanthine (d) oncell suspension CDPK activityroseus Tdc and Str Effect of phosphatase inhibitor and accumulation of group protector in UV-B-induced cultures of C (a), MBPK activity (b), Effect of phosphatase inhibitor and phosphatase thiol group protector on UV-B-induced CDPK activity (a), MBPK activity (b), Tdc and Str gene expression (c) and accumulation of catharanthine (d) in cell suspension. .. 44:675-685 Shilpa R, Phd thesis: Studies on UV-B-induced signal transduction events leading to catharanthine/ vindoline production in cell suspension cultures of Catharanthus roseus and cloning and characterization of the key enzyme – 3 – deoxy D – arabino-heptulosonate – 7- phosphate synthase gene involved in tryptophan biosynthesis Indian Institute of Science, Bangalore, India 2006 Bradford MM: A Rapid and... induced by variety of elicitors, such as yeast elicitor on tobacco [31,32], chitin oligosaccharides in tomato [33], fungal oligosaccharides in red clover roots [34], and fungal elicitors in spruce [35] and parsley cell suspensions [36] Using NAC, Ca2+ channel blocker and broad range of kinase inhibitor staurosporine, we showed that protein phosphorylation and an increase in intracellular calcium levels... subunit of the mammalian NADPH oxidase complex was shown to be responsive for ROS accumulation in response to bacterial protein elicitor harpin [38] It has been shown that protein phosphorylation is needed for the production of ROS in potato tubers, spruce and tobacco cells [39] The inhibitory effects of the protein kinase inhibitor staurosporine and Ca2+ channel blockers on UV-Binduced ROS production in. .. member of the MAPK family Our results (Figure 4) also suggest the involvement of Ca2+-dependent protein kinase (s) or Ca-CaM (calmodulin)-dependent protein kinase (s) in the UV-B response MAP kinases, members of a group of serine/threonine protein kinases are important transducers of intracellular signals via protein phosphorylation that is initiated by various extracellular stimuli, and they are involved... Hi-media Laboratories, India Catharanthine and vindoline were obtained from Shanghai kangai biologicals, China Staurosporine and suramin were obtained from MP Biomedicals, Germany Monoclonal antibodies to phospho-serine and phospho-tyrosine, complete protease inhibitor cocktail and myelin basic protein were purchased from Upstate laboratories, U.S.A SB 203580 (P38 inhibitor), PD 98059 (ERKK inhibitor) and... (1 M in water) 1 and 10 mM (1 M in water) 10 and 100 nM (10 µM in ethanol) 0.1 and 1 mM (0.1 M in water) 0.5 and 5 µM (0.5 mM in ethanol) 5 uM (0.5 mM in DMSO) Calcium chelator [56] Scavenger of reactive oxygen species and protects thiol group of phosphatases from inactivation [26] Inhibitor of serine-threonine phosphatases [27] Inhibitor of tyrosine phosphatases [26] Broad range inhibitor of serine-threonine... accumulation of catharanthine were all inhibited by suramin Suramin per se is not known to affect medium alkalinization directly but acts via a receptor [17] This suggested that suramin acts upstream of the afore-mentioned UV-B-induced responses and the UV-B-induced TIA biosynthesis The inhibitory effect of suramin on the UV-B responses supports role of a putative cell surface receptor in UV-B signal... quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 1976, 72:248-254 Putnam-Evans CL, Harmon AC, Cormier MJ: Purification and characterization of a novel calcium-dependent protein kinase from soybean Biochem 1990, 29:2488-2495 Stratmann JW, Ryan CA: Myelin basic protein kinase activity in tomato leaves is induced systemically by wounding and increases in. .. inhibitor of serine-threonine kinases Inhibits binding of growth factors to their receptors [17] L-type calcium channel blocker [28, 29] ERKK inhibitor [58] 70 nM (7 µM in DMSO) P38 MAPK inhibitor [58] 40 nM (4 µM in DMSO) JNK inhibitor [59] Page 14 of 17 (page number not for citation purposes) BMC Plant Biology 2007, 7:61 sonicated (30 % amplitude, 15 pulses) in a buffer containing 50 mM HEPES-KOH pH . possible involvement of these kinases in UV-B signaling pathway leading to catharanthine accumulation. The C. roseus cells were treated with inhibitors of protein kinases and the UV-B-induced. and catharanthine content were examined. Staurosporine, a potent inhibitor of serine-threonine kinases, SB 203580, an inhibitor of P38 class of MAP kinase, PD 98059, an inhibitor ERKK class of. Studies on UV-B-induced signal transduc- tion events leading to catharanthine/ vindoline production in cell suspension cultures of Catharanthus roseus and cloning and characterization of the key